Enzyme immunoassay for antibodies in serum using a covalent chromatographic method for separation of the bound label.
A sensitive enzyme immunoassay system for the measurement of antibodies in serum was developed by the use of a separation method based on the thiol-disulfide interchange reaction. Serum samples including antibodies (anti-insulin, anti-human chorionic gonadotropin, or anti-beta-D-galactosidase) were incubated with antigens labeled with beta-D-galactosidase from Escherichia coli. Each reaction mixture was then passed through a small column (0.1 ml) of (anti-IgG)IgG-Sepharose 4B, in which the (anti-IgG)IgG had been coupled by means of a disulfide bond. The column was washed to remove the unbound label, then the antibody-bound label was eluted from the column with a buffer containing 25 mM dithiothreitol, which cleaved the disulfide bonds between the Sepharose matrix and the anti-IgG antibody molecules. By measuring the enzyme activity in the eluate, the amount of antibodies could be determined even in a serum sample which contained antibodies corresponding to as little as 0.01% of the standard antisera. Preliminary experiments showed that the present method can be used to detect the anti-insulin antibody in the sera from insulin-treated diabetic patients.
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000941:Antigens; D003920:Diabetes Mellitus / Q000276:immunology; D004220:Disulfides; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G; D007330:Insulin Antibodies / Q000032:analysis; D011817:Rabbits / Q000276:immunology; D012016:Reference Values; D013438:Sulfhydryl Compounds; D001616:beta-Galactosidase
D000906:Antibodies; D000941:Antigens; D004220:Disulfides; D007074:Immunoglobulin G; D007330:Insulin Antibodies; D013438:Sulfhydryl Compounds; D001616:beta-Galactosidase